Stress can be defined as an environmental transient challenging homeostasis. Most organisms have developed "stress responses" that buffer the stress or attenuate its effects. Perhaps the best known and least understood stress response in mammals is "activation" of the hypothalamic-pituitary-adrenal (HPA) axis. Like all neuroendocrine systems, the HPA axis has feedback loops to maintain homeostasis. Under non-stressful (basal) or mild stress, pituitary ACTH secretion is halted at a certain threshold determined by the saturation of steroid receptors binding to circulating corticosteroids. ACTH release is restored when the corticosteroid concentration drops below the threshold. Under both these conditions, increased ACTH secretion can be attenuated by administration of dexamethasone. In contrast, in severe stress, both CRH and vasopressin stimulate the secretion of ACTH. Under these conditions, ACTH secretion cannot be attenuated with dexamethasone, demonstrating an abrogation of this ACTH/corticosteroid feedback loop. Morphine, however, effectively inhibits ACTH release suggesting this potent analgesic is mimicking an alternative feedback loop controlled by an endogenous opioid. One candidate is pituitary beta-endorphin which is co- synthesized from the same prohormone (POMC) and co-secreted with ACTH. Gene transfer studies in heterologous cells show that the proteolytic processing of POMC to ACTH and b-endorphin can be accounted for largely by the actions of the neuroendocrine-specific endoproteases, PC2 and PC3. The goal of this proposal is to confirm the proposed role of these two endoproteases in the maturation of POMC in pituitary cell cultures and use this information to selectively manipulate the synthesis of mouse pituitary beta-endorphin in transgenic animals. This technology will then be used to test the hypothesis that pituitary beta-endorphin modulates ACTH secretion in severe stress. The pituitary endorphin- deficient mice will then be studied to assess the role of the opioid peptide in the response to specific physiologic and psychologic stressors. To attain this goal; (i) The hypothesis that PC3 is responsible for specific cleavages of POMC in pituitary cells will be confirmed by attenuating PC2 expression in AtT-20 cells as well as primary cultures of anterior lobe and neurointermediate lobe pituicytes using both antisense nucleic acids and active site directed peptides, (ii) Amino acid residues in the prohormone which determine the cleavage site sequence specificity of PC2- and PC3-specific cleavage will be identified in heterologous cells as well as pituitary cell cultures. Residues critical for PC2-cleavage site specificity will be incorporated into the cleavage site of alpha1-PI Pittsburgh to generate a PC-specific serpin which will be used in PC2 "knockout" studies, (iii) PC2 antisense RNA or a PC2-specific serpin will be expressed in transgenic mice employing the POMC promoter to direct pituitary-specific expression. Pituitaries from transgenic animals will be isolated and the effect of the PC2 inhibition on the synthesis of POMC peptides will be examined and, (iv) Examine the effect of deficiency of pituitary beta-endorphin on the response to specific physiologic stressors.